Primer Design

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Visual OMP’s primer design capabilities are a breakthrough in how oligos are designed for assays. In any single design, we can account for amplicon size, primer lengths, Tm, template secondary structure, hairpin folding, primer dimers, mishybridization, crosshybridization (when using multiplex) and checks using BLAST.

By enabling users to find quick solutions using lab tested default parameters or to completely customize parameters to a user’s unique technology, complete and accurate solutions can be solved for even the most complex situation. How can a software application solve such complex issues, such as a 10 plex multiplex designs? When necessary, all advanced parameters, such as specific cutoff values for mishybridization and target secondary structure are made fully accessible and customizable. In this way, we put the control back in the users hands, to be able to design primers for even the most unique situation.

We have devised a logical mathematical way of representing the needs of the scientific user. Let’s think about a typical statement by someone trying to design a simple PCR primer:

“I want to design a pair of primers, with the amplicon length around 100, but no less than 50 and no more than 150 nucleotides. I would like the primers to be no shorter than 15 nucleotides, and no longer than 25 nucleotides, but more importantly, I would like their respective Tms to be around 65. I suppose I could go as low as 60 degrees, but I’d really like the Tm to be around 65, higher is not a problem.”

Visual OMP is able to easily solve this kind of request by turning verbal ideas into mathematical models. By setting minimum, maximum and optimal values as well as bonus and penalty weights for deviating from the optimum, it is possible to describe how to treat any single parameter. By combining relative parameters with each other, we can prioritize different aspects of a user’s need into a single cohesive design.

How can this system apply to something like an amplicon length? This kind of mathematical model enables Visual OMP to design around a specifically declared amplification window (area to be amplified) or to automatically select a region based on user based choices, such as low target secondary structure, low mishybridization, and other thermodynamics. Primers can also be weighted or penalized the closer or farther they are to an amplification window. In this way, it is possible for Visual OMP to translate a verbal request into a hierarchical list of solutions based on the user’s input. In fact, Visual OMP even allows you to force it to create an amplicon of a specific length.

Primers can also be independently designed giving the right and left primers different characteristics. Probably the most noteworthy case is that of Single Base Extension (SBE) experiments where primers must be designed as a specific location. Visual OMP allows users to input a single nucleotide, with a fixed 5’ or 3’ end such that the primers are designed at an exact base.

When performing multiplex experiments or experiments in which primers could bind to a third party sequence, Visual OMP can prescreen all sequences beforehand. When Visual OMP designs primers, all possible solutions can be checked against any user-defined sequences. And if sequences are too long, it’s possible to ask Visual OMP to use a BLAST algorithm to check any possible solution against any database or genome, even if that database is user defined. Visual OMP takes care to ensure that solutions that are outputted are of the highest quality so that they work, the first time out.